CN115389690B - Comprehensive identification method for benzotriazole ultraviolet absorber pollutants in environment - Google Patents
Comprehensive identification method for benzotriazole ultraviolet absorber pollutants in environment Download PDFInfo
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Abstract
A comprehensive identification method of benzotriazole ultraviolet absorber pollutants in environment comprises the steps of obtaining a suspected target analysis database of benzotriazole ultraviolet absorber pollutants; carrying out liquid chromatography-mass spectrometry analysis on an environmental sample to be detected to obtain data-dependent acquisition data and data-independent acquisition data; performing matching analysis on the data-dependent acquisition data based on the information of the suspected target compound, and determining the structure of the compound matched with the suspected target compound; extracting characteristic fragment ions of the benzotriazole ultraviolet absorber type contaminant from the data-independent acquisition data to extract candidate compounds based on the characteristic fragment ions; and analyzing the chromatographic information and the mass spectrum information related to the candidate compound in the data-dependent acquisition data to determine the structure of the candidate compound. The invention realizes the comprehensive identification of benzotriazole ultraviolet absorber pollutants in the environment, can be applied to various complex environment media, and has wide application prospect.
Description
Technical Field
The invention relates to the field of environmental analysis chemistry, in particular to a comprehensive identification method of benzotriazole ultraviolet absorber pollutants in the environment.
Background
Benzotriazole ultraviolet absorbers (Benzotriazole UV absorbers, BZT-UVs) are artificially synthesized organic compounds, and can absorb full spectrum ultraviolet rays in natural light, so that the benzotriazole ultraviolet absorbers can be widely used as chemical additives in the fields of plastics, coatings, textiles, paint, printing and dyeing, building materials, cosmetics and the like. During industrial production and daily use, BZT-UVs inevitably enter the environmental medium and are enriched in organisms. The environmental distribution and toxic effects of BZT-UVs have received widespread attention. The known BZT-UVs in the current environment are extremely limited in variety, and it is important to comprehensively identify occurrence conditions of the BZT-UVs in an environment medium and accurately evaluate health hazards and ecological risks of the BZT-UVs.
The analysis method is the key for comprehensively identifying BZT-UVs. Traditionally, BZT-UVs environmental monitoring adopts a targeting analysis method based on low-resolution mass spectrum, relies on a real standard, and is difficult to realize screening and identification of unknown BZT-UVs. The popularization and application of the high-resolution mass spectrum provide a technical means for comprehensively identifying BZT-UVs in a complex environment medium. The suspected targeted and non-targeted analysis method based on high-resolution mass spectrum is one of effective strategies for guiding the comprehensive identification of BZT-UVs homologs. However, how to screen and identify the structure of the collected mass high-resolution mass spectrum data based on the suspected targeted and non-targeted analysis methods is a technical problem to be solved.
Disclosure of Invention
It is therefore a primary object of the present invention to provide a method for the comprehensive identification of BZT-UVs type contaminants in an environment, in order to at least partially solve at least one of the above mentioned technical problems.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a comprehensive identification method of BZT-UVs pollutants in an environment comprises the following steps: obtaining a suspected targeted analysis database of BZT-UVs pollutants, wherein the suspected targeted analysis database is constructed to store structural information and mass spectrum prediction information of suspected targeted compounds; carrying out liquid chromatography-mass spectrometry on an environmental sample to be detected, wherein the collection of fragment ions in the mass spectrometry is respectively carried out in a data-dependent collection mode and a data-independent collection mode so as to obtain data-dependent collection data and data-independent collection data of the environmental sample to be detected; performing matching analysis on the data-dependent acquisition data based on the structural information and the mass spectrum prediction information of the suspected target compound, and determining or possibly configuring a compound matched with the suspected target compound in the environmental sample to be detected; extracting characteristic fragment ions of BZT-UVs type contaminants from the data independent acquisition data to extract candidate compounds based on the characteristic fragment ions, wherein the candidate compounds are distinguished from compounds that match the suspected targeting compound; and analyzing chromatographic information and mass spectrum information related to the candidate compound in the data-dependent acquisition data to determine the possible structure of the candidate compound.
Based on the technical scheme, the comprehensive identification method of BZT-UVs pollutants in the environment has at least one or a part of the following beneficial effects:
the invention carries out suspected target analysis on data-dependent acquisition data in liquid chromatograph-mass spectrum analysis data of an environmental sample to be detected based on a constructed BZT-UVs pollutant suspected target analysis database to obtain a determined or possible structure of a compound matched with a suspected target compound, carries out non-target analysis on data-independent acquisition data to obtain a possible structure of a candidate compound different from the suspected target compound, realizes comprehensive identification and structural identification of BZT-UVs possibly existing in the environmental sample to be detected through complementation of the two identification methods of the suspected target analysis and the non-target analysis, does not depend on a real standard substance, can be implemented under the condition of lacking any reference compound information, and can be applied to complex environmental media to provide technical support for environmental monitoring.
Drawings
Fig. 1 is a flow chart of a method for comprehensively identifying BZT-UVs type pollutants in an environment of the invention.
Fig. 2 is a detailed flow chart of the overall identification method of BZT-UVs type contaminants in the environment of example 1 of the present invention.
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.
In the process of realizing the invention, the technical difficulty of identifying BZT-UVs by adopting a suspected target analysis strategy is found how to pertinently establish a suspected target analysis database in the suspected target analysis process; the non-targeted analysis does not have any reference compound information, and the application difficulty is that the collected mass high-resolution mass spectrum data are screened and structurally identified. The invention realizes comprehensive identification of BZT-UVs homologs in complex environment medium by combining a suspected targeting analysis and a non-targeting analysis complementary identification method based on the structural characteristics of BZT-UVs. It should be noted that the suspected targeting database refers to a database containing known BZT-UVs compounds that may exist in the environment, and the known BZT-UVs compounds that may exist in the environment are suspected targeting compounds.
Specifically, according to some embodiments of the present invention, a method for comprehensive identification of BZT-UVs type contaminants in an environment is provided, comprising the following steps A-E (FIG. 1).
Step A: and obtaining a suspected targeted analysis database of BZT-UVs pollutants, wherein the suspected targeted analysis database is constructed to store structural information and mass spectrum prediction information of the suspected targeted compound.
According to an embodiment of the present invention, the suspected targeted analysis database in this step is constructed by the following steps A1 to A3.
In step A1, all compounds having a 2-hydroxybenzotriazole structural fragment are screened from the public chemical library.
According to embodiments of the present invention, the disclosed chemical data includes, but is not limited to, the China existing chemical List (IECSC), the U.S. toxic substance control method List (TSCA), the European Union chemical registration, assessment, authorization and restriction List (REACH), the Canadian national Material List (DSL), and the like.
In order to facilitate accurate screening of compounds, according to an embodiment of the present invention, the step A1 specifically includes: calculating the matching degree, namely the similarity, of the compound to be screened in the public chemical database and the SMILES of the 2-hydroxybenzotriazole based on a valley coefficient algorithm; and determining the compound to be screened as a compound with a 2-hydroxybenzotriazole structural fragment under the condition that the matching degree meets the preset condition.
Further alternatively, the molecular structural formula C of the 2-hydroxybenzotriazole is calculated by using a Python platform and a valley coefficient algorithm 12 H 9 N 3 O, which corresponds to the SMILES formula c1=cc=c (C (=c1) n2n=c3c=cc=cc3=n2) O. SMILES is a technique in the art for describing chemical structures by character stringsThereby converting the complex chemical structural formula into a computer-recognizable character string form.
As a preferred example, in the case where the degree of matching is not less than 0.7, the compound to be screened corresponding to the degree of matching is determined as a compound having a 2-hydroxybenzotriazole structural fragment. Further preferably, after the computer screening based on the foregoing matching degree calculation, further manual inspection is performed to improve the accuracy.
In step A2, the biological conversion products of the screening compounds in the organism are predicted based on the metabolic conversion pathways of phase I, phase II and phase III in the organism.
According to an embodiment of the invention, the metabolic conversion pathway is determined based on the metabolic conversion pathway and BZT-UVs molecular structural characteristics of the reported contaminant in the organism, for example involving oxidation, hydrolysis, reduction, methylation, sulfation, acetylation, and various binding reactions, etc.
For example, BZT-UVs can be predicted in vivo phase I and phase II metabolic transformation products using the Compound Discoverer (version 3.3) software of Thermo Fisher Inc.
In step A3, a suspected targeted assay database is constructed based on the screened compounds and the corresponding bioconversion products.
It will be appreciated that the screening compound and the corresponding bioconversion product are used as the suspected targeting compound, and the suspected targeting assay database contains structural information and mass spectrometry prediction information for the screening compound and the corresponding bioconversion product.
According to an embodiment of the invention, the structural information includes a compound name and a chemical formula, and the mass spectrum prediction information includes an adduct ion exact mass number and predicted secondary fragment ion information. Alternatively, the compound name may be, for example, chemical name full scale, chemical name short, or the like; the chemical formula may be, for example, a formula such as C 13 H 11 N 3 O, etc.; the exact mass number of the adduct ions may be, for example, the exact mass number of sodium addition, the exact mass number of protons addition, etc., and the exact mass number of protons addition is more commonly used; the predicted secondary fragment ion information may be predicted, for example, using MetFrag software.
And (B) step (B): and carrying out liquid chromatography-mass spectrometry on the environmental sample to be detected, wherein the collection of fragment ions in the mass spectrometry is respectively carried out in a data-dependent collection mode and a data-independent collection mode so as to obtain data-dependent collection data and data-independent collection data of the environmental sample to be detected.
According to the embodiment of the invention, in the step B, an ultra-high liquid chromatograph and high-resolution mass spectrometry combined system can be used for carrying out liquid chromatograph-mass spectrometry analysis on the environmental sample to be detected, so as to be beneficial to analysis and detection on the complex environmental sample.
According to an embodiment of the invention, the liquid chromatography conditions are configured as a gradient elution procedure to better separate the environmental sample to be tested.
According to the embodiment of the invention, the data-dependent acquisition mode of mass spectrometry is a primary ion scanning mode and a secondary ion scanning mode under the positive ion condition of an atmospheric pressure chemical ionization source, an electrospray ionization source or an atmospheric pressure optical ionization source; the data independent acquisition mode IS a primary ion scanning mode under the condition of adopting an atmospheric pressure chemical ionization source, an electrospray ionization source or an atmospheric pressure optical ionization source positive ions in an in-source collision induced dissociation (IS-CID for short) mass spectrum mode. The ionization sources used in the data independent acquisition mode and the data dependent acquisition mode can be the same or different, and compared with the data dependent acquisition data, the data independent acquisition data adopts a full spectrum breaking mode in the source so as to acquire fragment information of all compounds, thereby being beneficial to comprehensive identification of BZT-UVs pollutants.
Further, the data-dependent acquisition data includes a primary mass spectrum (MS 1 ) And a secondary Mass Spectrum (MS) 2 )，MS 1 Obtained by an electrostatic field orbitrap, the scanning range is mass-to-charge ratio m/z=100-1000, ms 1 For obtaining information such as the mass number of the adduct ions, MS 2 Acquired by high energy collision dissociation (HCD) mode, the scan range is MS dependent 1 The mass-to-charge ratio m/z of the parent ion is mainly used for acquiring secondary fragment ion information.
According to an embodiment of the present invention, the data dependent acquisition data requires high resolution mass spectrometry data deconvolution analysis, and the signal intensity threshold may be, for example, 10e 4.
Further, the data independent acquisition data includes an MS containing information of all fragment ions 1 ，MS 1 Obtained by an electrostatic field orbitrap, the scanning range is mass-to-charge ratio m/z=100-1000, and is mainly used for acquiring the signal intensity of characteristic fragment ions and the molecular formulas of all candidate compounds.
Step C: and carrying out matching analysis on the data-dependent acquisition data based on the structural information and the mass spectrum prediction information of the suspected target compound, and determining or possibly determining the structure of the compound matched with the suspected target compound in the environmental sample to be detected.
According to an embodiment of the present invention, the step C is a suspected targeting analysis, and the determining or possible structure of the precursor compound is obtained by analyzing whether the precursor compound matching the suspected targeting compound exists in the environmental sample to be tested, which specifically includes sub-steps C1 to C3.
In sub-step C1, matching the mass number of the addition ions in the data-dependent acquisition data with the accurate mass number of the addition ions of the suspected target compound, and screening the matched suspected precursor compound from the suspected target compound;
in sub-step C2, matching the secondary fragment ions in the data-dependent acquisition data with predicted secondary fragment ion information of the suspected precursor compound, and screening the suspected precursor compound to obtain a matched precursor compound;
in sub-step C3, the identity or possible structure of the precursor compound is determined based on the chromatographic retention behavior of the precursor compound and the secondary fragment ions, the precursor compound being a compound that matches the suspected targeting compound.
Through the substeps C1 to C3, the mass number of the adduct ions and the secondary fragment ions are sequentially matched, which is favorable for more accurately determining whether the sample to be detected contains the suspected target compound in the suspected target database, and then the determination or possible structure of the precursor compound is proposed through analyzing the chromatographic retention behavior (such as chromatographic retention time and the like) of the compound with successfully matched characteristics and the mass spectrum fragmentation law determined by the secondary fragment ions.
Step D: extracting characteristic fragment ions of BZT-UVs type contaminants from the data-independent acquisition data to extract candidate compounds based on the characteristic fragment ions, wherein the candidate compounds are distinguished from compounds that match the suspected targeting compound.
According to the embodiment of the invention, the step D is non-targeted analysis, the characteristic fragment ions of BZT-UVs pollutants are usually secondary fragment ions shared by the BZT-UVs pollutants, so that all BZT-UVs pollutants in an environmental sample to be detected can be determined by reverse calculation based on the characteristic fragment ions, meanwhile, only candidate compounds which are different from the precursor compounds in the step C are extracted and subjected to subsequent step analysis in the step D, and the suspected targeted analysis and the non-targeted analysis are coupled, so that the comprehensive identification of the BZT-UVs pollutants is realized while the analysis process is simplified.
According to an embodiment of the invention, this step D comprises in particular the sub-steps D1 to D3.
In sub-step D1, an ion-flow diagram (EIC) containing characteristic fragment ions of BZT-UVs type contaminants is extracted from the data-independent acquisition data.
In sub-step D2, from the MS corresponding to EIC retention time 1 And extracting candidate molecular formulas corresponding to the characteristic fragment ions.
In sub-step D3, a candidate molecular formula that is distinguished from the precursor compound is determined as the molecular formula of the candidate compound.
Through the above sub-steps D1 to D3, the molecular formula of the candidate compound to which it may correspond, e.g. C, is found based on the extracted characteristic fragment ions 20 H 25 N 3 O 2 And then step E is re-entered to infer its likely structure based on mass spectrometry data.
According to embodiments of the invention, the characteristic fragment ions of BZT-UVs type contaminants may include known broadly detected BZT-UVs secondary fragment ions, e.g. [ C 6 H 6 N 3 ] + (m/z＝120.0562) And [ C ] 12 H 10 N 3 O] + (m/z= 212.0824) and may also include characteristic fragment ions of the invention obtained by experimental analysis, e.g. [ C ] 13 H 10 N 3 O] + (m/z= 224.0824) and [ C 15 H 14 N 3 O] + (m/z＝252.1137)。
Step E: and analyzing the chromatographic information and the mass spectrum information related to the candidate compound in the data-dependent acquisition data to determine the possible structure of the candidate compound.
According to an embodiment of the invention, this step E specifically comprises obtaining chromatographic retention behavior and secondary fragment ions of the candidate compound from the data-dependent acquisition data to determine the possible structure of the candidate compound.
More specifically, the chromatographic retention behavior and the secondary fragment ion of the candidate compound are obtained from the data-dependent acquisition data based on the molecular formula of the candidate compound in the step D, and the reasonable candidate compound is identified by analyzing the chromatographic retention behavior and the mass spectrum fragmentation rule based on the secondary fragment ion, so as to infer the possible structure of the candidate compound.
According to an embodiment of the invention, the method of the invention further comprises determining fragment ions characteristic of BZT-UVs type contaminants, in particular comprising the following steps F and G.
Step F: and performing liquid chromatography-mass spectrometry on a plurality of real standard substances existing in the environment in the suspected targeted analysis database in an IS-CID mass spectrometry mode. As a preferred embodiment, the real standard may be selected by text mining, including but not limited to keyword extraction, etc., from BZT-UVs that have been found in the environment.
Step G: and adjusting different cone hole voltages to analyze characteristic fragment ion signals of a plurality of real standard substances so as to determine the optimal cone hole voltages and the characteristic fragment ions of corresponding BZT-UVs pollutants. It is understood that the characteristic fragment ions of BZT-UVs type contaminants are secondary fragment ions common to a plurality of authentic standards.
According to an embodiment of the present invention, the data independent acquisition mode in step B is preferably performed under the optimal cone voltage condition.
According to the embodiment of the invention, the acquisition of the real standard substance can be used for accurately determining the characteristic fragment ions of BZT-UVs pollutants so as to accurately perform non-targeted analysis, and can be used for performing targeted analysis on the compound matched with the real standard substance in the environment to be detected based on the real standard substance.
Further, the method of the invention also comprises the targeted analysis of the environmental sample to be detected, and specifically comprises the following steps: and carrying out matching analysis on the data-dependent acquisition data based on chromatographic retention behaviors and mass spectrum information of the plurality of real standard substances, and determining the target compounds and the content matched with the plurality of real standard substances. The quantitative analysis of the target compound in the environmental sample to be detected is realized through the target analysis.
According to the embodiment of the invention, the method can be applied to various complex environment media, and the environment sample to be detected comprises water samples, solid samples and biological samples containing BZT-UVs; the water sample can be industrial sewage, water inlet and outlet of a sewage treatment plant, river water, surface water, sea water, drinking water, underground water and the like, the solid sample can be bottom mud of the sewage treatment plant, water body sludge, sediment, soil, indoor dust, atmospheric particulates and the like, and the biological sample can be human breast milk, urine, serum, animal organs, fish, birds, sharks, mollusks, plants and the like.
The following describes the technical scheme of the invention in detail by listing a plurality of specific embodiments. It should be noted that the following specific embodiments are only examples and are not intended to limit the present invention.
In the following examples, some reagents and detection instruments are described as follows:
reagent: the 12 BZT-UVs authentic standard are listed in table 1 below.
Liquid chromatograph-mass spectrometer: ultra performance liquid chromatography and high resolution mass spectrometry (Ultimate-3000 liquid chromatography-Orbitrap high resolution mass spectrometry, thermo Fisher, USA); chromatographic column: waters ACQUITY C18 (1.7 μm, 2.1X100 mm).
Example 1
This example is a laboratory test example, focusing on the implementation of the identification of BZT-UVs present in a labeled sample using a set-up methodology, comprising the specific steps of (fig. 2):
step one: summarizing the 16 BZT-UVs found in the environment as shown in table 1 by a text mining mode, and selecting 12 BZT-UVs with higher detection rate and detection concentration to purchase a real standard.
TABLE 1
Step two: based on a public chemical database containing IECSC, TSCA, REACH and DSL, calculating the structural similarity between a compound to be screened and 2-hydroxybenzotriazole in the public chemical database by using a Python platform and a valley coefficient algorithm, and setting a score threshold value to be 0.7, namely determining the compound to be screened with the similarity being more than or equal to 0.7 as the compound with the 2-hydroxybenzotriazole structural fragment. Since the authentic standard falls within the range of compounds screened in this step, this step was additionally screened for 21 BZT-UVs homologs that did not purchase the authentic standard.
Step three: based on the known 30 in vivo contaminant metabolic conversion pathways shown in Table 2, BZT-UVs were predicted in vivo as phase I and phase II metabolic conversion products using the software Compound Discoverer (version 3.3) from Thermo Fisher Inc. The established pathway encompasses all 20 BZT-UVs bioconversion products currently known.
TABLE 2
Step four: and (3) constructing the BZT-UVs homologues obtained in the steps one to three into a suspected target analysis database (i.e. an in-house database) which comprises the compound name, the chemical formula, the precise mass number of the adduct ions and the information of the secondary fragment ions predicted by MetFrag software.
Step five: and (3) continuously injecting the BZT-UVs with 12 real standard substances selected in the step (I) in a needle pump mode under an IS-CID mass spectrum mode, adjusting the cone hole voltages to be 10, 20, 30, 40, 50, 60 and 70eV respectively, and comparing the characteristic fragment ion signal intensities of different BZT-UVs under the serial cone hole voltages.
Preferably 30eV IS the optimum cone aperture voltage for scanning the characteristic fragment ions in IS-CID mass spectrometry mode to meet that all characteristic fragment ions have the appropriate signal intensity.
Step six: 1mL of a methanol solution containing 12 real standards was prepared at a concentration of 100. Mu.g/L, and then analyzed by liquid chromatography-mass spectrometry.
Liquid chromatography conditions: the temperature of the chromatographic column is 35 ℃; the mobile phase consisted of a methanol solution (A) containing 0.5mM ammonium acetate and an aqueous solution (B) containing 0.5mM ammonium acetate. The mobile phase gradient elution procedure was: firstly, 70% A is kept for 1min; increasing A to 100% within 14 min; then 100% A for 5min; then decrease a to 70% in 0.1 min; last 70% a for 4.9min; the flow rate of the mobile phase is 0.3mL/min; the sample volume was 5. Mu.L.
Mass spectrometry conditions: the ion source adopts an atmospheric pressure chemical ionization source positive ion mode; spray voltage 3500V; the temperature of the ion source is 200 ℃, the temperature of the ion transmission tube is 350 ℃, and the atomization temperature is 400 ℃; sheath gas, purge gas and assist gas pressures were 35, 1 and 10Arb, respectively. Data dependent acquisition mode parameters: MS (MS) 1 Obtained by means of an electrostatic field orbital trap, resolution is 120000 (m/z=200), scan range is m/z=100-1000, maximum injection time is 100ms, automatic gain control target is 3e6, s-lens RF is 60%. MS (MS) 2 Resolution 60000 (m/z=200), collision energy was set at 10, 30, 50%, MS by HCD mode acquisition 1 Parent ion isolation by quaternary leverThe isolation window width m/z=1, the fragment ions are detected by the electrostatic field orbitrap, and the scan range depends on the parent ion m/z. The data independent acquisition mode adopts an IS-CID mass spectrum mode, and the parameters are as follows: MS (MS) 1 Obtained by an electrostatic field orbital trap, resolution is 120000 (m/z=200), scan range is m/z=100-1000, maximum injection time is 100ms, automatic gain control target is 3e6, s-lens RF is 60%, mass range is normal, and cone voltage is 30eV.
Step seven: and D, performing suspected target analysis on the data of the data-dependent acquisition mode in the step six in Compound Discoverer (version 3.3), and identifying BZT-UVs and predicting bioconversion products recorded in the list. The flow is as follows: 1, deconvolution of high-resolution mass spectrum data, and a signal intensity threshold 10e4;2, matching a suspected target database, namely matching two mass spectrum data, namely matching the mass number of the added ions of the primary mass spectrum and matching the secondary fragment ions, wherein in the primary mass spectrum matching, the mass spectrum deviation is 5ppm, the isotope threshold is 75%, and the signal to noise ratio is 5;3, analyzing the chromatographic retention behavior and mass spectrum fragmentation rules of the precursor compound with the successfully matched characteristics, and providing a definite or possible structure.
Step eight: non-targeted analysis of step six data independent acquisition mode data was performed in Xcalibur Qual Browser (version 4.0) software to identify BZT-UVs not recorded in the chemical industry manifest, not belonging to the predicted bioconversion products, industrial intermediates or impurity classes. The flow is as follows: 1, extracting 4 characteristic fragment ions, i.e. [ C ] 6 H 6 N 3 ] + (m/z＝120.0562)、[C 12 H 10 N 3 O] + (m/z＝212.0824)、[C 13 H 10 N 3 O] + (m/2= 224.0824) and [ C 15 H 14 N 3 O] + An EIC of (m/z= 252.1137); 2, according to EIC diagram retention time, from corresponding MS 1 Molecular formula of candidate compound corresponding to the characteristic fragment ion is distinguished from precursor compound successfully matched in the step seven; 3, analyzing chromatographic retention behavior and mass spectrum fragmentation rule of candidate compounds under data-dependent acquisition mode based on molecular formula, matching reasonable candidates based on mass spectrum data, and providingA deterministic or possible structure is derived.
It can be appreciated that, further, the BZT-UVs type pollutants identified in the steps seven and eight can be semi-quantitatively analyzed by means of a structural similarity standard; and can also carry out the target analysis to the environmental sample to be tested based on a plurality of real standard substances, and in particular, in this embodiment, because the sample to be tested is the real standard substance, the target analysis process is not repeated.
Through the specific steps, all the 12 BZT-UVs in the standard substance solution are identified, the accuracy is 100%, the reliability of the identification method is shown, and the BZT-UVs in the sample can be accurately identified. The seven suspected target analysis and the eight non-target analysis realize correct identification of BZT-UVs in the sample, and the two identification methods are complementary, so that comprehensive identification of BZT-UVs in the environment can be ensured.
Example 2
Similar to the implementation of example 1, the addition of the standard methanol solution was replaced with a standard real environmental sediment sample, and the purpose of this example was to test the ability of the method to identify BZT-UVs in environmental samples in the presence of matrix interference.
The procedure is substantially as in example 1, except that in step six, the actual environmental sample is subjected to pretreatment including procedures of accelerated solvent extraction, gel permeation chromatography purification, silica gel column purification and rotary evaporation nitrogen blowing reconstitution. The results show that 12 marked BZT-UVs are all identified, and the method is applicable to complex environment media and has little interference effect by a matrix.
Example 3
The purpose of this example is to test the overall recognition capability of the method for BZT-UVs in environmental samples. The collected environmental sample is a chlamys farreri biological sample.
The procedure is substantially as in example 1, except that in step six, the chlamys farreri sample is subjected to pretreatment including procedures of accelerated solvent extraction, gel permeation chromatography purification, silica gel column purification and rotary nitrogen blowing reconstitution. The results show that 21 BZT-UVs, including 10 targeted BZT-UVs (UV-P, UV-PS, UV-234, UV-320, UV-326) were identified in commonUV-327, UV-328, UV-329, UV-350 and UV-360), 5 bioconversion products (structure shown in formula I-V) and 4 impurity BZT-UVs (structure shown in formula 1-7). Wherein dechlorinated and methylated BZT-UVs bioconversion products (UV-326-H and UV-327-CH) 3 ) And impurity BZT-UVs are found in an environment medium for the first time through the identification method, so that the method can comprehensively identify the BZT-UVs in the environment and fill the technical blank in the field of the existing BZT-UVs environment analysis chemistry.
Formula I-formula V: determination or possible structure of the identified BZT-UVs bioconversion products
BZT@m/z＝322：
BZT@m/z＝330：
BZT@m/z＝340：
BZT@m/z＝410：
Formulas 1 to 7: determination or possible structure of identified impurity BZT-UVs
Example 4
The purpose of this example was to test the high throughput recognition capability of the method for BZT-UVs in large-scale environmental samples. The environmental samples collected were 129 mollusk samples covering 9 cities (daLian, yingkou, hu island, north Daihe river, tianjin, shouguang, suaeda, fulai, yantai and Weihai) in the Bohai sea region of China.
The procedure is substantially as in example 1, except that in step six, the mollusc sample is subjected to a pretreatment including an accelerated solvent extraction, gel permeation chromatography purification, silica gel column purification and spin-on nitrogen-blow reconstitution procedure. The whole recognition system is used for completing analysis of all environmental samples within 24 hours, and the recognized BZT-UVs with real standard substances are verified by the standard substances, so that the method can accurately, rapidly and high-flux complete comprehensive recognition of the BZT-UVs in the environment.
The comprehensive results of the above embodiments show that the comprehensive recognition method of BZT-UVs pollutants in the environment is high in accuracy, and the suspected targeted analysis method and the non-targeted analysis method are complementary; the method can be applied to various complex environment media, and has little interference effect by the matrix; the identified compounds are comprehensive in types, and known, unknown and conversion products can be identified with high efficiency; the method has the characteristics of high speed and high flux, and can finish large-scale environmental sample identification in a short time. Therefore, the method provided by the invention has universal applicability and wide application prospect.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.
Claims (9)
1. A method for the comprehensive identification of benzotriazole uv absorber-based contaminants in an environment comprising the steps of:
acquiring a suspected targeted analysis database of benzotriazole ultraviolet absorber pollutants, wherein the suspected targeted analysis database is constructed to store structural information and mass spectrum prediction information of suspected targeted compounds;
carrying out liquid chromatography-mass spectrometry on an environmental sample to be detected, wherein the collection of fragment ions in the mass spectrometry is respectively carried out in a data-dependent collection mode and a data-independent collection mode so as to obtain data-dependent collection data and data-independent collection data of the environmental sample to be detected;
performing matching analysis on the data-dependent acquisition data based on the structural information and the mass spectrum prediction information of the suspected target compound, and determining or possibly configuring a compound matched with the suspected target compound in the environmental sample to be detected;
extracting characteristic fragment ions of a benzotriazole ultraviolet absorber type contaminant from the data independent acquisition data to extract candidate compounds based on the characteristic fragment ions, wherein the candidate compounds are distinguished from compounds that match the suspected targeting compound;
analyzing chromatographic information and mass spectrum information related to the candidate compound in the data-dependent acquisition data to determine a possible structure of the candidate compound;
the suspected targeted analysis database is constructed by the following steps:
screening all compounds having 2-hydroxybenzotriazole structural fragments from the public chemical library;
predicting bioconversion products of the selected compounds in the organism based on the metabolic conversion pathways of phase I, phase II and phase III in the organism;
the suspected targeted assay database is constructed based on the screening compound and the corresponding bioconversion product.
2. The comprehensive identification method according to claim 1, wherein the screening all compounds having 2-hydroxybenzotriazole structural fragments from the public chemical database comprises:
calculating the matching degree of the SMILES type of the compound to be screened and the 2-hydroxybenzotriazole in the public chemical database based on a valley coefficient algorithm;
and determining the compound to be screened as a compound with a 2-hydroxybenzotriazole structural fragment under the condition that the matching degree meets a preset condition.
3. The comprehensive identification method according to claim 1, wherein the data-dependent acquisition mode is a primary ion scanning and secondary ion scanning mode under positive ion conditions using an atmospheric pressure chemical ionization source, an electrospray ionization source or an atmospheric pressure optical ionization source;
the data independent acquisition mode is a primary ion scanning mode under the condition of positive ions in an in-source collision induced dissociation mass spectrum mode.
4. The comprehensive identification method according to claim 1, wherein the structural information includes a compound name and a chemical formula, and the mass spectrum prediction information includes an addition ion exact mass number and predicted secondary fragment ion information.
5. The method of claim 4, wherein the performing a matching analysis on the data-dependent acquisition data based on the structure information and the mass spectrometry prediction information of the suspected targeted compound, and determining a determination or possible structure of a compound in the environmental sample to be tested that matches the suspected targeted compound comprises:
matching the mass number of the addition ions in the data-dependent acquisition data with the accurate mass number of the addition ions of the suspected target compound, and screening the suspected target compound to obtain a matched suspected precursor compound;
matching the secondary fragment ions in the data-dependent acquisition data with the predicted secondary fragment ion information of the suspected precursor compounds, and screening the suspected precursor compounds to obtain matched precursor compounds;
determining the identity or possible structure of the precursor compound based on the chromatographic retention behavior and secondary fragment ions of the precursor compound, the precursor compound being a compound that matches the suspected targeting compound.
6. The method of claim 5, wherein extracting characteristic fragment ions of benzotriazole ultraviolet absorber type contaminants from the data independent acquisition data to extract candidate compounds based on the characteristic fragment ions comprises:
extracting an ion flow graph containing characteristic fragment ions of benzotriazole ultraviolet absorber type contaminants from the data independent acquisition data;
extracting candidate molecular formulas corresponding to the characteristic fragment ions from a primary mass spectrum corresponding to the ion flow diagram retention time;
a candidate molecular formula that is distinguishable from the precursor compound is determined as the molecular formula of the candidate compound.
7. The method of claim 1, wherein analyzing chromatographic information and mass spectrometry information associated with the candidate compound in the data-dependent acquisition data to determine a likely structure of the candidate compound comprises:
the chromatographic retention behavior and secondary fragment ions of the candidate compound are obtained from the data-dependent acquisition data to determine the likely structure of the candidate compound.
8. The comprehensive identification method according to claim 1, further comprising:
performing liquid chromatography-mass spectrometry on a plurality of real standard substances existing in the environment in the suspected targeted analysis database in an in-source collision induced dissociation mass spectrometry mode;
and adjusting different cone hole voltages to analyze characteristic fragment ion signals of the plurality of real standard substances so as to determine the optimal cone hole voltages and the corresponding characteristic fragment ions of the benzotriazole ultraviolet absorber pollutants.
9. The comprehensive identification method of claim 8, further comprising:
and carrying out matching analysis on the data-dependent acquisition data based on chromatographic retention behaviors and mass spectrum information of the plurality of real standard substances, and determining the target compounds and the content matched with the plurality of real standard substances.
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